34] Direct photoaffinity labeling with cyclic nucleotides

Abstract

In direct photoaffinity labeling technique UV irradiation of binding proteins in the presence of their radioactive ligand results in covalent attachment of the two moieties. This method obviates the possibility that a chemically modified ligand might bind with insufficient affinity and specificity, or in a topological orientation incongruent with that of the native ligand. In addition, it avoids the complexities of synthesizing a modified ligand, with attendant problems of yield and purification. Direct photoaffinity labeling is an extremely simple technique. Many criteria for the specificity of labeling can rapidly be tested. Labeled receptors can be analyzed by a number of standard methods at various levels of resolution. This chapter describes a procedure for using unmodified radioactive cyclic nucleotides as direct photoaffinity labels for high-affinity macromolecular receptors in tissue extracts. By UV irradiation of tissue extracts and [ 3 H] cAMP or [ 3 H] cGMP, one can achieve specific covalent labeling of a class of peptides that bind cyclic nucleotides with high affinity. This photoreaction to label cyclic ucleotide binding sites in messenger ribonucleoprotein-like particles has been used. Several other applications of direct photoaffinity labeling with cyclic nucleotides have recently appeared. These include the labeling of renal plasma membrane receptors and of binding proteins in rat liver and hepatoma subcellular compartments.